www.nature.com/scientificreports OPEN Responses to chemical cross‑talk between the Mycobacterium ulcerans toxin, mycolactone, and Staphylococcus aureus Laxmi Dhungel1, Lindsey Burcham1, Joo Youn Park1, Harshini Devi Sampathkumar1, Albert Cudjoe2, Keun Seok Seo1 & Heather Jordan1* Buruli ulcer is a neglected tropical disease caused by the environmental pathogen, Mycobacterium ulcerans whose major virulence factor is mycolactone, a lipid cytotoxic molecule. Buruli ulcer has high morbidity, particularly in rural West Africa where the disease is endemic. Data have shown that infected lesions of Buruli ulcer patients can be colonized by quorum sensing bacteria such as Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa, but without typical pathology associated with those pathogens’ colonization. M. ulcerans pathogenesis may not only be an individual act but may also be dependent on synergistic or antagonistic mechanisms within a polymicrobial network. Furthermore, co‑colonization by these pathogens may promote delayed wound healing, especially after the initiation of antibiotic therapy. Hence, it is important to understand the interaction of M. ulcerans with other bacteria encountered during skin infection. We added mycolactone to S. aureus and incubated for 3, 6 and 24 h. At each timepoint, S. aureus growth and hemolytic activity was measured, and RNA was isolated to measure virulence gene expression through qPCR and RNASeq analyses. Results showed that mycolactone reduced S. aureus hemolytic activity, suppressed hla promoter activity, and attenuated virulence genes, but did not afect S. aureus growth. RNASeq data showed mycolactone greatly impacted S. aureus metabolism. These data are relevant and signifcant as mycolactone and S. aureus sensing and response at the transcriptional, translational and regulation levels will provide insight into biological mechanisms of interspecifc interactions that may play a role in regulation of responses such as efects between M. ulcerans, mycolactone, and S. aureus virulence that will be useful for treatment and prevention. Buruli ulcer (BU) disease is a necrotizing skin disease whose etiological agent is Mycobacterium ulcerans (MU). Te major virulence factor of MU is mycolactone, a cytotoxic macrocyclic lipid encoded by a giant plasmid pMUM001 that was acquired during emergence from an M. marinum progenitor1. Mycolactone leads to detach- ment and death of fbroblasts, epithelial cells, endothelial cells and karatinocytes, following exposure of 24–48 h, and leads to local and systemic immunomodulation2. Mycolactone’s mechanism on host cells has been shown to profoundly inhibit several host cytokines and chemokines that are dependent on Sec61 mediated translocation such as IL-17, TNFα, IL-6, chemokines IL-8 and MIP2, and adhesion molecules such as L-selectin. But other cytokines such as IL-1α, chemokines CCCL1, CXCL5 and adhesion molecules such as P-selectin, E-selectin, intracellular adhesion molecule (ICAM1) and lymphocyte function-associated antigen 1 are less afected3,4. Also, Interleukin-1β (IL-1β), a Sec-independent cytokine released through the caspase activated pathway has been shown to be less inhibited by mycolactone, suggesting selective inhibition of infammatory cytokines in BU disease3,5,6. In fact, a recent study showed that mycolactone induced IL-1β production by murine and human macrophages, possibly through mycolactone-mediated membrane disturbance and the production of reactive oxygen species that trigger NLRP3/1 infammasome activation, leading to the release of active IL-1β7. And, mycolactone does not appear to directly afect neutrophils in BU disease as its toxicity toward neutrophils is observed only in high doses, and absence of neutrophils in BU wounds is mainly attributed to poor neutrophil chemotaxis toward MU3,8. 1Department of Biological Sciences, Mississippi State University, P.O. Box GY, Starkville, MS 39762, USA. 2Georgia State University, Atlanta, GA, USA. *email: [email protected] Scientifc Reports | (2021) 11:11746 | https://doi.org/10.1038/s41598-021-89177-5 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. (A) Dose efect of mycolactone on S. aureus growth measured by OD600 for up to 16 h. (B) Comparison of OD600 of S. aureus incubated alone, or with mycolactone or EtOH at 3H, 6H, and 24H. Staphylococcus aureus, S. epidermidis and Pseudomonas aeruginosa have been isolated from the infected lesions of BU patients9–12. However, disease pathology normally associated with these pathogens, such as pain, pus, or redness is strikingly absent in these ulcers, especially prior to MU antibiotic treatment 9,10,13. Despite the lack of pathology, co-colonization by these pathogens may promote delayed wound healing, especially afer the initiation of MU antibiotic therapy. Staphylococcus aureus is a primary cause of skin and sof tissue infections, prosthetic-joint infections and infective endocarditis 14–17, but may also be human normal fora, particularly resid- ing within anterior nares 18. S. aureus expresses several factors required for colonization (adhesins) and invasion (coagulase, staphylokinase)19, lysis (hemolysins), immune evasion (leukotoxins), and increased pathogenicity (enterotoxins and TSST1), which are regulated by the SaeRS (S. aureus exoprotein expression) two component system (among others)20–22; but also by the accessory gene regulator system (Agr), that regulates virulence through quorum sensing mechanisms 23,24. What is less clear, then, is whether the absence of pus and other pathology in a co-infected wound from a BU patient is due to decreased virulence of S. aureus or immune suppression caused by mycolactone. S. aureus skin infection is typically characterized by abscess formation mediated by neutrophils, where the absence of pro- infammatory cytokine IL-1β has been associated with increased lesion size in murine skin infections4,25. Also, S. aureus colonizing BU wounds contains virulence genes such as hla (hemolysin), sak (staphylokinase), and luk (several leukocidins including lukD, lukE, lukF, lukS, and PVL) with a potential to cause serious and invasive infections. However, absence of any pus or invasive infection in BU patients colonized with S. aureus suggests attenuation of S. aureus virulence in BU disease and a potential role of mycolactone in driving this attenuation 9,26. Te macrocyclic structure of mycolactone is similar to quorum sensing (QS) compounds in other bacteria. Studies have shown that some QS molecules such as 3-oxo-C12-Homoserine lactone produced by P. aeruginosa can inhibit QS systems of other bacteria, such as inhibition of S. aureus sarA and agr expression27. Auto-inducing peptides produced by S. epidermidis and S. schleiferi can also inhibit expression of Agr regulated virulence genes in S. aureus20,28. Te inability of isolated pathogens such as S. aureus to cause disease in BU wounds is intriguing and suggest mechanisms of MU to attenuate the virulence of those pathogens during infection, contributing to niche competition, and predominance of MU in the wound. On the other hand, co-colonization in a MU wound could allow S. aureus nutrient acquisition and a means of dispersal at a lowered energy expense. Terefore, the purpose of this study was to understand the efect of mycolactone on S. aureus growth, expression of virulence factors and hemolysis. Results S. aureus growth is not inhibited by mycolactone. Staphylococcus aureus was incubated with myco- lactone (concentration 0 ng–10 µg) over time to determine whether there was any efect by mycolactone on S. aureus growth. Based on optical density (OD600nm), low to moderate mycolactone concentrations (1 ng–1 µg) had no efect on S. aureus growth for up to 16H, though 10 µg mycolactone had some efect on S. aureus growth afer 7H (Fig. 1A). We also measured corresponding S. aureus growth in association with our gene expression experiments across timepoints. Tere was no statistical diference in growth between S. aureus containing 0, 50, 100 and 300 ng/mL mycolactone and control at 3H, 6H and 24H timepoints (Fig. 1B). Mycolactone reduces S. aureus hemolytic activity. Staphylococcus aureus hemolytic activity was sig- nifcantly decreased for S. aureus incubated with mycolactone (concentration 50, 100 and 300 ng) compared to S. aureus incubated with the mycolactone vehicle control (EtOH) at 3H. While hemolysis was decreased for S. aureus incubated with all concentrations at 6H compared to controls, this decrease was not statistically signif- cant. At 24H, hemolysis was only signifcantly reduced for S. aureus incubated with 300 ng mycolactone (Fig. 2). Mycolactone leads to modulation of S. aureus global regulator gene expression. Te efect of mycolactone on S. aureus agrA, saeR and hla virulence genes was determined by RT-qPCR. Te results showed that agrA was not signifcantly modulated at 3H (Fig. 3A) but was signifcantly downregulated for S. aureus incubated with mycolactone (300 ng at 6H (p = 0.043, Fig. 3B). However, agrA returned to control levels at 24H Scientifc Reports | (2021) 11:11746 | https://doi.org/10.1038/s41598-021-89177-5 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 2. Mycolactone co-Incubation reduced S. aureus hemolysis over time. Graph showing OD500 of red blood cells incubated with water (positive control), S. aureus alone, S. aureus plus 50 ng mycolactone, S. aureus plus 100 ng mycolactone, or S. aureus plus 300 ng mycolactone. Signifcance was determined as < 0.05. (a–c), and (d,e) denotes signifcantly diferent